Spontaneous Integration of Transmembrane Peptides into a Bacterial

An antimicrobial peptide, temporin L, and its derivative (TL-A2) were employed as anchor peptides and displayed streptavidin on a bacterial magnetic p...
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Anal. Chem. 2004, 76, 3764-3769

Spontaneous Integration of Transmembrane Peptides into a Bacterial Magnetic Particle Membrane and Its Application to Display of Useful Proteins Tsuyoshi Tanaka, Hajime Takeda, Yoriko Kokuryu, and Tadashi Matsunaga*

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo, Japan

An antimicrobial peptide, temporin L, and its derivative (TL-A2) were employed as anchor peptides and displayed streptavidin on a bacterial magnetic particle (BMP) membrane. The ribotoxin L3 loop (L3) and the arginine-chain peptide (R12), which are carrier peptides permeable to eukaryotic cell membranes, were also used. The peptides were labeled with a fluorescent dye, 4-fluoro-7-nitrobenzofurazan (NBD), at the N-terminal region (NBD-peptides) and mixed with BMPs. A specific integration of NBDtemporin L into a BMP membrane was observed. The basic amino acids in temporin L played an important role in the integration into BMPs. Biotin conjugated to the N-terminus of temporin L was integrated into a BMP membrane. The C-terminus of temporin L was incorporated into a BMP membrane, and the N-terminus was located on the BMP membrane surface. The present study shows that temporin L is a stable molecular anchor on BMPs by the binding of soluble protein to the N-terminus. Peptides that are inserted into eukaryotic and prokaryotic membranes have been successfully used as antibiotics and carriers of proteins1,2 and DNA.3 A number of candidate peptide carriers into a eukaryotic cell, such as the human immunodeficiency virus type 1 (HIV-1) Tat, Antennapedia (ANTP),4 or ribotoxin L3 loop (L3) peptides,5 have been reported. Much attention has been paid to HIV Tat (48-60) as an arginine-rich peptide with highly efficient translocation.3 Arginine-chain peptides were also internalized into macrophage cells.6 Peptides using antimicrobial activity against prokaryotes can be spontaneously inserted into cell membranes. These peptides vary in structure (R-helix or β-sheet; cyclic or linear), length (cecropin A: 37 amino acids,7 magainins * Corresponding author. Phone: +81-42-388-7020. Fax: +81-42-385-7713. E-mail: [email protected]. (1) Fawell, S.; Seery, J.; Daikh, Y.; Moore, C.; Chen, L. L.; Pepinsky, B.; Barsoum, J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 664-668. (2) Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. Science 1999, 285, 1569-1572. (3) Schwarze, S. R.; Dowdy, S. F. Trends Pharmacol. Sci. 2000, 21, 45-48. (4) Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1994, 269, 10444-10450. (5) Langedijk, J. P. J. Biol. Chem. 2002, 277, 5308-5314. (6) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. J. Biol. Chem. 2001, 276, 5836-5840. (7) Steiner, H.; Hultmark, D.; Engstrom, A.; Bennich, H.; Boman, H. G. Nature 1981, 292, 246-248.

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2: 23 amino acids,8 temporin L: 13 amino acids9), and orientation. Cationic linear peptides such as magainin 2 and temporin L can form amphiphilic structures with the hydrophobic part organized in a helix when associated with membranes. These characteristics enable us to use the peptide as carriers and anchors of cell membranes. Bacterial magnetite (Fe3O4) particles (BMPs) are 50-100 nm in size, covered with a lipid bilayer membrane.10,11 The lipid bilayer imparts BMPs with better dispersion qualities compared with artificial magnetic particles. Superior dispersion permits various applications of BMPs. Antibody,12,13 enzyme,14 and receptor protein have been fixed on BMPs and applied to immunoassay,12,13 mRNA isolation techniques,15 and DNA detection.16-18 BMPs are easily purified from a culture of magnetic bacteria by magnetic separation using a permanent magnet. The BMP membranes, which are probably derived from the invagination of the cytoplasmic membrane, are similarly composed of a lipid bilayer and associated proteins. MagA was identified as an integral iron translocating protein and isolated in a recent analysis of membrane proteins for the elucidation of the mechanisms of BMP synthesis in M. magneticum AMB-1.19-22 MagA has been used as an anchor (8) Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5449-5453. (9) Rinaldi, A. C.; Mangoni, M. L.; Rufo, A.; Luzi, C.; Barra, D.; Zhao, H.; Kinnunen, P. K.; Bozzi, A.; Di Giulio, A.; Simmaco, M. Biochem. J. 2002, 368, 91-100. (10) Nakamura, N.; Hashimoto, K.; Matsunaga, T. Anal. Chem. 1991, 63, 268272. (11) Nakamura, N.; Matsunaga, T. Anal. Chim. Acta 1993, 281, 585-589. (12) Matsunaga, T.; Kawasaki, M.; Yu, X.; Tsujimura, N.; Nakamura, N. Anal. Chem. 1996, 68, 3551-3554. (13) Tanaka, T.; Matsunaga, T. Anal. Chem. 2000, 72, 3518-3522. (14) Matsunaga, T.; Togo, H.; Kikuchi, T.; Tanaka, T. Biotechnol. Bioeng. 2000, 70, 704-709. (15) Sode, K.; Kudo, S.; Sakaguchi, T.; Nakamura, N.; Matsunaga, T. Biotechnol. Tech. 1993, 7, 688-694. (16) Matsunaga, T.; Nakayama, H.; Okochi, M.; Takeyama, H. Biotechnol. Bioeng. 2001, 73, 400-405. (17) Yoshino, T.; Tanaka, T.; Takeyama, H.; Matsunaga, T. Biosens. Bioelectron. 2003, 18, 661-666. (18) Nakayama, H.; Arakaki, A.; Maruyama, K.; Takeyama, H.; Matsunaga, T. Biotechnol. Bioeng. 2003, 84, 96-102. (19) Nakamura, C.; Burgess, J. G.; Sode, K.; Matsunaga, T. J. Biol. Chem. 1995, 270, 28392-28396. (20) Matsunaga, T.; Tsujimura, N.; Okamura, Y.; Takeyama, H. Biochem. Biophys. Res. Commun. 2000, 268, 932-937. (21) Okamura, Y.; Takeyama, H.; Matsunaga, T. J. Biol. Chem. 2001, 276, 48183-48188. (22) Arakaki, A.; Webb, J.; Matsunaga, T. J. Biol. Chem. 2003, 278, 8745-8750. 10.1021/ac035361m CCC: $27.50

© 2004 American Chemical Society Published on Web 05/20/2004

Table 1. Amino Acid Sequences of Peptides Used in This Study peptide name

residues

temporin L TL-A2 biotin-N-temporin L-lysine temporin L-lysine-biotin ribotoxin L3 loop (L3) poly arginine (R12)

13 13 14 14 13 12

amino acid sequence

pI

N-LIRGLFKSFWQVF 11.1 N-LIAGLFASFWQVF 5.6 biotin-LIRGLFKSFWQVFK N-LIRGLFKSFWQVFK-biotin N-KLIKGRTPIKFGK 11.4 N-RRRRRRRRRRRR 13.1

protein for the expression of firefly luciferase23 and protein A13 on a BMP membrane. The artificial integration onto BMPs was successfully achieved using MagA-luciferase complexes extracted from recombinant Escherichia coli membranes.24 The artificial integration of useful protein was more efficient as compared with previously reported gene fusion techniques and is a promising method for a highly efficient assembling of soluble proteins on BMPs. However, this method is time-consuming and inefficient because solubilization of membrane proteins using detergents and integration of MagA fusion proteins into a BMP membrane by sonication are difficult. In this study, an efficient integration of peptides spontaneously into a BMP membrane was investigated for establishing a novel protein assembling technique. Cationic peptides, R12 and L3, and an antimicrobial peptide, temporin L, have been employed as anchor peptides onto BMPs. Temporin L showed highly efficient integration into BMPs than R12 and L3. Furthermore, biotin-labeled peptides have been used in order to assemble streptavidin on BMPs. MATERIALS AND METHODS Materials. Sulfosuccinimidy1-6′-(biotinamido)-6-hexanamido hexanoate (Sulfo-NHS-LC-LC-biotin) and sulfosuccinimidyl 6-(3′[2-pyridyldithio]-propionamido)hexanoate (Sulfo-LC-SPDP) were purchased from Pierce (Rockford, IL). AlexaFluor350-labeled streptavidin (AlexaFluor350-streptavidin) was purchased from Molecular Probes Inc. (Eugene, OR). N-[(Dimethylamino)-1H1,2,3-triazoid[4,5,-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide and Fmoc-L-amino acid-OH, Fmoc-L-amino acid-PEG-PS used for Fmoc chemistry, were purchased from Applied Biosystems (Lincoln Centre Drive Foster City, CA). Piperidine[hexahydropyridine] was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). 4-Fluoro-7-nitrobenzofurazan (NBD) was purchased from Dojindo Laboratories (Kumamoto, Japan). Phospholipase D (PLD) from Streptomyces sp., phosphatidic acid (PA), and phosphatidylethanolamine (PE) were purchased from Sigma Chemical Co. (St. Louis, MO). 2-Aminoethanol was obtained from Wako Pure Chemicals Industries Ltd. (Osaka, Japan). Other reagents were of analytical-reagent or laboratory grade. Deionized distilled water was used in all procedures. Peptide Synthesis. Table 1 shows the peptides used in this study. Temporin L, with antimicrobial activity against Grampositive and Gram-negative bacteria, and its derivative, temporin L-A2 (TL-A2), were prepared as model peptides. Temporin L to reverse its direction (II) was used in this study, although both (23) Nakamura, C.; Kikuchi, T.; Burgess, J. G.; Matsunaga, T. J. Biochem. (Tokyo) 1995, 118, 23-27. (24) Matsunaga, T.; Arakaki, A.; Takahoko, M. Biotechnol. Bioeng. 2002, 77, 614-618.

temporin L in the natural form (I) and temporin L (II) interact with the lipid membrane. TL-A2, in which alanine was substituted in positions 3 and 7 from the amino terminal of temporin L, reduced the electrostatic interactions between peptide and membrane. A lysine residue was added in the C-terminus of temporin L (temporin L-lysine), to conjugate biotin in the C-terminus using Sulfo-NHS-LC-LC-biotin. Sulfo-NHS-LC-LC-biotin was reacted with an amino group in the side chain of lysine. Biotin-N-temporin L-lysine was prepared to conjugate biotin to the N-terminus of temporin L-lysine. Membrane permeable peptides, arginine-rich (R12),6 and ribotoxin L3 loop (L3)5 were also synthesized. R12 (12 arginine residues) has a translocation activity similar to Tat (4860). L3 is positioned in the C-terminus of pestivirus envelope glycoprotein. All peptides were synthesized by a standard Fmoc-based solidphase method with a peptide synthesizer PSSM-8 (Shimadzu Corp., Kyoto, Japan) and analyzed by mass spectrometry, LCQ DECA XP (Thermo Electron Corp., Chicago, IL). Deprotection of the N-terminus protecting group (Fmoc) was achieved by 30% piperidine in dimethylformamide. The side chain amine of the lysine and the arginine is protected with a Boc group and a pbf group, respectively, that are not removed until the peptide is cleaved from the resin support. A fluorescent dye, 4-fluoro-7nitrobenzofurazan (NBD), was labeled with their respective N-terminus. Biotin was coupled as its N-hydroxysuccinimidyl ester to the N-terminus of temporin L-lysine. Subsequently, the Boc in the lysine side chain and the pbf in the arginine side chain were deprotected by trifluoroacetic acid (TFA) including 2.5% (v/v) m-cresol, 7.5% (v/v) ethanedithiol, and 7.5% (v/v) thioanisole. To conjugate biotin to the C-terminus of temporin L-K, 4-methyltrytyl (Mtt) protected-lysine was assembled at the C-terminus. After removal of MTT by dichloromethane including 1% (w/v) TFA and 1-5% (w/v) triisopropylsilane and subsequent conjugation of biotin to the side chain amine, Fmoc at the N-terminus was removed by piperidine treatment. Peptide solutions were prepared using double-distilled deionized water. Sequence analysis software, Protean (Lasergene, DNASTAR Inc., Madison, WI), was used to predict the secondary structure and pI of peptides. The system provided a means of studying the theoretical structure of each peptide, such as polar/apolar orientation, space fill models, and charge distribution. Purification of BMPs from Magnetospirillum magneticum AMB-1. The magnetic bacterium M. magneticum AMB-1 was grown in modified magneto spirillirum growth medium (MSGM)25 at room temperature under microaerobic conditions for 7-10 days. The stationary phase cells were centrifuged at 10000g for 10 min at 4 °C, resuspended in phosphate buffered saline (10 mM PBS, pH 7.4), and disrupted by three passes through a French press at 1500 kg/cm2 (Ohtake Works Co. Ltd., Tokyo). BMPs were magnetically isolated from disrupted cell fractions using a neodymium-boron (Nd-B) magnet. The BMPs were washed 10 times with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4) and HEPES buffer containing 1 M sodium chloride to remove membrane surface proteins freely attached onto BMPs by electrostatic interactions. Purified BMPs were boiled at 100 °C for 10 min in (25) Blakemore, R. P.; Maratea, D.; Wolfe, R. S. J. Bacteriol. 1979, 140, 720729.

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1 N NaOH solution to investigate the protein contents. The supernatant was used to determine the membrane protein content on BMP according to the Lowry method.26 Modification of a BMP Membrane by Proteinase K and PLD Treatment. Phospholipids mainly consisting of phosphatidylethanolamine (PE) contribute to the negative charge of BMP surface, in which the zeta potential of BMP surface is -25 mV at pH 7.0.13 Purified BMPs were treated with proteinase K to remove the membrane proteins. BMPs (1 mg) were suspended with 1 units proteinase K in HEPES buffer (10 mM, pH 8.7) for 30 min at room temperature with pulsed sonication (1 time pulses at 10min intervals). The BMPs (1 mg) were separated magnetically from the reaction mixture using a Nd-B magnet and washed three times with HEPES buffer (10 mM, pH 7.4). To modify the characteristics of BMP surface, purified BMPs were further treated with PLD, which has a high transphosphatidylation activity27,28 to generate phosphatidic acid (PA) from PE. BMPs were suspended with 50 units/mL of PLD in 10 mM Tris buffer containing 150 mM sodium chloride and 100 µM CaCl2 for 30 min at room temperature with pulsed sonication. The BMPs were again suspended in the PLD solution in the presence of 2-aminoethanol to reproduce PE from PA. The phospholipid on BMPs was extracted by a mixture of chloroform/methanol (2:1) as described by Kates29 and finally dissolved with chloroform. The phospholipid fraction was analyzed by thin-layer chromatography (TLC) using a silica gel plate (silica gel 60F254; Merck, Tokyo, Japan) with chloroform-methanol-water (65:25:4, v/v/v) as development solvent and detected by Dittmer reagent.30 PLD treatment results in the release of 2-aminoethanol from PE based on hydrolysis reaction. The remaining amino groups on the BMP membrane were measured using Sulfo-LC-SPDP. Sulfo-LC-SPDP contains amine reactive N-hydroxysuccinimide residue and pyridyl disulfide residue. Sulfo-LC-SPDP (1.05 mg) was added in 1 mL of BMP suspension (1 mg/mL) to react with the amino groups on BMPs. After incubation at room temperature for 30 min, modified BMPs were magnetically separated from the reaction mixture using a Nd-B magnet and washed three times with 1.0 mL of PBS. The Sulfo-LC-SPDP-modified BMPs were dispersed in 0.5 mL of 20 mM dithiothreitol (DTT) in PBS and incubated with sonication for 30 min at room temperature. The absorbance of released pyridylthiol in the supernatant was measured by a spectrophotometer at 343 nm. Peptide Integration into a BMP Membrane. BMPs (350 µg) were mixed with 1 mL of NBD-peptide solutions (30 µM) in HEPES buffer at room temperature. After incubation for 0.5 or 1 h, the BMPs associated with peptides were washed five times with HEPES or PBS containing various sodium chloride concentrations. The integration of peptides to BMPs was evaluated by fluorescent microscopy and measurement of fluorescence intensity. Fluores(26) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 245, 4508. (27) Dittrich, N.; Ulbrich-Hofmann, R. Biotechnol. Appl. Biochem. 2001, 34, 189194. (28) Stieglitz, K. A.; Seaton, B. A.; Roberts, M. F. Biochemistry 2001, 40, 1395413963. (29) Kates, M. In Laboratory techniques in biochemistry and molecular biology; Work, T. S., Work, E., Eds.; North-Holland Publ.: Amsterdam, 1972; Vol. 3, Part II, pp 270-610. (30) Shaw, B. R.; Corden, J. L.; Sahasrabuddhe, C. G.; Van Holde, K. E. Biochem. Biophys. Res. Commun. 1974, 61, 1193-1198.

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cence intensity (ex: 464 nm, em: 512 nm) of BMPs (350 µg/ mL) was measured using a fluorescent microplate reader (FLUOstar Optima, Moritex Corp., Tokyo, Japan). Streptavidin Binding to Biotin-Labeled Temporin L Integrated into a BMP Membrane. The orientation of temporin L in the membrane was evaluated by the binding of AlexaFluor 350labeled streptavidin (AlexaFluor 350-streptavidin) to biotinylated peptide on BMPs. Biotin conjugated to the N-terminus or the C-terminus of temporin L-lysine was added to BMPs suspension (350 µg/mL) and incubated for 30 min at room temperature. The integration of the peptides into BMPs was performed as described above. After washing with PBS (pH 7.4) containing 1 M sodium chloride, the BMPs associated with biotin-labeled peptides were reacted with 200 µg/mL of AlexaFluor 350-streptavidin for 30 min at room temperature. Fluorescence intensity (ex: 345 nm, em: 440 nm) of BMPs (350 µg/mL) was measured using a fluorescent microtiter plate reader. Biotin chemically conjugated to BMPs was also prepared using Sulfo-NHS-LC-LC-biotin as a control. Purified BMPs (2 mg) were washed once with 10 mM NaOH (1 mL) and three times with 50 mM sodium borate buffer containing 150 mM sodium chloride (pH 8.5). The BMPs were mixed with 0.5 mg of Sulfo-NHS-LCLC-biotin in sodium borate buffer and incubated for 1 h with pulsed sonication (2 min sonication 8 min interval). After washing with 10 mM phosphate buffer containing 1 mM EDTA, 150 mM sodium chloride, and 0.1% Tween 20 (PBST, pH 7.4), 100 µg of AlexaFluor 350-streptavidin was reacted with modified BMPs in Tris buffer containing 1 mM EDTA and 1 M sodium chloride for 1 h. After washing three times with PBST, the fluorescence of AlexaFluor 350- streptavidin binding to biotinylated BMPs was measured. Streptavidin-Assembly Using Complexes with AlexaFluor 350-Streptavidin and Biotin-Temporin L-Lysine into a BMP Membrane. To evaluate the utility of temporin L as a molecular anchor, a complex of AlexaFluor 350-streptavidin and biotin conjugated to the N-terminus of temporin L-lysine (biotin-Ntemporin L-lysine) was prepared and then mixed with 350 µg/ mL of BMPs. Biotin-N-temporin L-lysine (30 µM) was reacted with 100 µg/mL of AlexaFluor 350-streptavidin. After incubation of the complexes with BMPs for 30 min at room temperature, the BMPs were washed with HEPES buffer containing 1 M NaCl. RESULTS Integration of Various Peptides into a BMP Membrane. The integration of various peptides including temporin L, TL-A2, L3, and R12 into a BMP membrane was evaluated by fluorescence of NBD conjugated to peptides as shown in Figure 1. The fluorescence by NBD-L3 and R12 attaching to BMPs was completely removed by washing five times with HEPES containing 1 M sodium chloride. The attaching of NBD-L3 and R12 is based on electrostatic interactions between cationic amino acids and negatively charged BMPs, but they are not integrated into the BMP membrane. L3 and R12 have translocation activity against a eukaryotic cell membrane, although the mechanisms still remain unknown. Therefore, L3 and R12 were not integrated into the BMP membrane which originated from a Gram-negative bacterium. NBD-temporin L showed the highest fluorescence. The fluorescence of NBD-TL-A2 on BMPs was repressed to 20% of temporin

Figure 1. Integration of various NBD-peptides into BMP membranes before and after proteinase K and PLD treatment. BMPs (350 mg/ mL) were mixed with 1 mL of 30 µM various NBD-peptides solution in HEPES buffer at room temperature, incubated for 1 h, and washed five times with PBS (pH 7.4) containing 1 M sodium chloride.

L. The basic amino acids in temporin L play an important role in the integration of the peptide into a BMP membrane. Use of Proteinase K and PLD-Treated BMP Membrane for the Integration of Various Peptides. The amount of protein in purified BMPs was 34 µg per 1 mg of BMPs. Eighteen micrograms of protein/mg BMPs, which corresponds to 53% of the BMP membrane proteins, was removed by proteinase K treatment. The remaining proteins (16 µg/mg BMPs) were incorporated into the BMP membrane. Correspondingly, the amino groups on the BMP membrane after the proteinase K treatment decreased to 50%. Proteinase K-treated BMPs were incubated with PLD to remove the amino groups of PE in the BMP membrane. As a result, the amino group of the surface of the BMP membrane decreased to only 9%. Integration of the NBDlabeled peptides in the proteinase K and PLD-treated BMP membrane is also shown in Figure 1. Similar results were obtained when using nontreated BMPs. However, a slight fluorescence was shown in L3 and R12 on proteinase K and PLD-treated BMPs. The surface of proteinase K and PLD-treated BMPs is more negatively charged than that of nontreated BMPs because of the lack of amino groups. The increase of a negative charge on BMPs might be the reason for the slight fluorescence observed when using cationic peptides possessing translocation activity. The binding of TL-A2 also increased when proteinase K and PLD-treated BMPs were used. In temporin L, only a slight decrease was observed in the fluorescence intensities by peptides integrated into proteinase K and PLD-treated and nontreated BMPs. However, NBDtemporin L showed still high fluorescence. Temporin L is useful as a molecular anchor integrated into the BMP membrane and used for further experiments. Effect of Sodium Chloride and pH on Integration of NBDTemporin L and R12 into a BMP Membrane. Subsequently, the effect of sodium chloride concentration and pH on integration of NBD-temporin L into BMPs was examined. NBD-R12 was used as a control. The fluorescence of NBD-temporin L or NBD-R12 on BMPs did not change upon changing the pH from 4.0 to 8.0 at a constant ionic strength condition, although the zeta potential for the BMPs changes from -2.5 to -25 mV in the range of pH from 4.0 to 7.0.13 The integration of NBD-R12 into a BMP membrane

Figure 2. Effect of sodium chloride concentrations on integration of NBD-temporin L and R12 into a BMP membrane. HEPES buffer (pH 7.4) was used for peptide binding assay. BMPs were resuspended in 10 mM HEPES buffer (pH 7.4) containing 500 mM sodium chloride after washing.

was not observed. Similar tendencies were obtained under various sodium chloride concentrations (Figure 2). No significant difference in fluorescence was observed under various sodium chloride concentrations when HEPES buffer containing 1 M sodium chloride was used as a washing buffer. These results suggest that pH ranged from 4.0 to 8.0, and sodium chloride up to 1 M did not have an affect on the integration of NBD-temporin L into BMPs. Washing of NBD-Temporin L and R12 Integrated into a BMP Membrane by HEPES Buffer Containing Various Concentrations of Sodium Chloride. The secondary structure of temporin L, which was confirmed by circular dichroism analysis, includes helical region and cationic residues in the peptide.31 The secondary structure of TL-A2, L3, and R12 as predicted by Protean showed an overall helical region, β-sheet, and turn structure, respectively. The pI values were 11.1 for temporin L, 5.6 for TLA2, 13.1 for R12, and 11.4 for L3 (Table 1). Temporin L, R12, and L3 are positively charged at neutral pH. Therefore, temporin L and R12 remaining in BMPs were investigated after washing with various sodium chloride concentrations in order to remove the binding with electrostatic interactions. HEPES buffer containing various sodium chloride concentrations was used as a washing buffer. Maximum fluorescences of NBD-temporin L and R12 on BMPs were obtained in the absence of sodium chloride (Figure 3a). The fluorescence was remarkably decreased as the sodium chloride concentration in the washing buffer was increased. No visible fluorescence of NBD-R12 on BMPs was confirmed by fluorescent microscopy after washing with HEPES buffer containing more than 100 mM sodium chloride, although a slight fluorescence was detectable by fluorescent microplate reader. The slight fluorescence (i.e. ∼10 fluorescence units) would be caused by the light scattering of BMPs in aqueous solutions as a background based on Mie scattering.18 By contrast, the fluorescence of NBD-temporin L on BMPs was observed even after washing in the presence of 1 M sodium chloride. The phenomenon indicates that R12 binds electrostatically to a BMP membrane, and temporin L was incorporated into a BMP membrane. To (31) Zhao, H.; Kinnunen, P. K. J. Biol. Chem. 2002, 277, 25170-25177.

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Figure 4. Phase-contrast (upper) and fluorescent (lower) microscopic photographs of peptide-binding BMPs with AlexaFluor 350streptavidin. (A) Temporin L-lysine-biotin LIRGLFKSFWQVFK-biotin. (B) Biotin-N-temporin L-lysine-biotin-LIRGLFKSFWQVFK. AlexaFluor350-streptavidin (200 µg/mL) was reacted with 350 µg/mL of BMPs for 30 min. Integration conditions: HEPES buffer (pH 7.4) for 30 min, washing conditions: PBS (pH 7.4) containing 1 M sodium chloride.

Figure 3. (a) Fluorescence intensities of NBD-temporin L and R12 integrated into a BMP membrane after washing with HEPES buffer (pH 7.4) containing various sodium chloride concentrations. BMPs were resuspended in HEPES buffer (pH 7.4) containing 500 mM sodium chloride after washing. (b) Effect of washing times on fluorescence intensities of NBD-temporin L and R12 integrated into a BMP membrane after washing with HEPES buffer (pH 7.4) containing 1 M sodium chloride. BMPs were resuspended in HEPES buffer containing 500 mM sodium chloride after washing.

eliminate the electrostatic binding, BMPs were washed several times under the same binding and washing conditions (Figure 3b). A stable fluorescence was obtained after washing four times. The fluorescence of NBD-temporin L on BMPs remained after washing 10 times. Binding of Streptavidin on Biotin-Temporin L Integated into a BMP Membrane. To assess the orientation of biotinlabeled temporin L integrated into a BMP membrane, the binding of AlexaFluor350-streptavidin to biotin-temporin L was investigated. In this experiment, lysine residue was added to the C-terminus of temporin L to immobilize biotin. The addition of lysine did not affect the peptide integration when NBD-labeled temporin L-lysine was used (data not shown). The fluorescence was observed on BMPs when AlexaFluor 350-streptavidin reacted with biotin binding to the N-terminal of temporin L-lysine (Figure 4). However, the BMP complex did not show the fluorescence when temporin L-lysine-biotin was employed. The C-terminus of temporin L is incorporated with the BMP membrane, and the N-terminus is located on the BMP membrane surface. To evaluate the utility of temporin L as an anchor in the complex form with streptavidin, the streptavidin-biotin complex using AlexaFluor 350-streptavidin and biotin-N-temporin L-lysine was first prepared 3768 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

Figure 5. Fluorescence intensities of AlexaFluor 350-streptavidin binding to biotinylated BMPs. 1: Biotin-N-temporin L-lysine integrated into BMPs. 2: Chemically conjugated to biotin on BMPs.

and then mixed with BMPs. The complex was also assembled on BMPs. The binding of AlexaFluor 350-streptavidin to biotin-Ntemporin L-lysine was almost similar to chemically conjugated biotin onto BMPs using Sulfo-LC-LC-biotin (Figure 5). These results indicate that temporin L is a stable molecular anchor on BMPs by the binding of soluble protein to the N-terminus of temporin L. DISCUSSION The present study shows that temporin L is a potential molecular anchor for displaying soluble proteins on BMPs. Temporin L was originally isolated from European red frog, Rana temporaria,32 which shows antimicrobial activity with lethal concentrations of 0.5-17.0 µM against Gram-positive and Gramnegative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.9 The calcein leakage from 50 µM phosphatidylcholine liposome was observed in the presence of 4 µM temporin L. In general, an antimicrobial mechanism entails (32) Simmaco, M.; Mignogna, G.; Canofeni, S.; Miele, R.; Mangoni, M. L.; Barra, D. Eur. J. Biochem. 1996, 242, 788-792.

membrane permeabilization and cell lysis by the interaction of peptides with the cytoplasmic membrane. In this study, 30 µM temporin L was added to BMPs for peptide integration. However, the BMP membrane was not disrupted in the presence of 30 µM temporin L. Since the membrane is attached on a magnetite surface, and the lipids are tightly packed on the magnetite, the BMP membrane was kept and BMPs aggregation did not occur. In a recent study, BMP membranes were shown to be probably derived from the cytoplasmic membrane.21 The integration was not affected by the change of pH and sodium chloride concentrations in buffers (Figure 2). This property offers an advantage for protein displaying on BMPs. Furthermore, the integration of temporin L was completed within 1 min. The result is consistent with a previous report, showing that 5 µM temporin L changes membrane permeability of liposomes in less than 4 min.9 Therefore, temporin L will be a powerful tool that allows a quick and spontaneous assembling of proteins. Assembling of enzymes, antibodies, and receptor proteins using an anchor peptide is the next target for future studies. The integration conditions for biomolecules have to be further improved to keep their optimum activity. Useful proteins fused to temporin L may be prepared by gene fusion experiments using host cells, such as E. coli and Bacillus sp. However, the expression will lead to the cell damage or death due to the antimicrobial activity. Therefore, in vitro translation will be preferred for the expression in host cells. Furthermore, the investigation whether the peptide acts as an anchor in the form of fusion protein should be examined in future works. Specific interactions of antimicrobial peptides against Grampositive bacteria and Gram-negative bacteria have been studied, but the exact mechanisms remain unknown. BMPs as stable magnetoliposomes provide novel and excellent model systems to analyze the integration mechanisms of antimicrobial peptides into the cytoplasmic membrane in Gram-negative bacteria. BMPs are more uniform in size (50-100 nm) and shape compared to

artificial magnetite particles and can be easily separated simply through the use of a magnet. In this study, PLD treatment was performed, leading to the conversion of PE to PA with the modification not significantly affecting the peptide integration. The conversion of PE to PA was confirmed by TLC. The membrane fraction of purified BMPs mainly contained PE. After PLD treatment, the main band in TLC was shifted to PA. Furthermore, PE was reproduced by PLD treatment in the presence of 2-aminoethanol. These results show that the PE on the BMP membrane was converted to PA by PLD treatment. Further modificaion of BMPs, such as conversion of PE to phosphatidyl choline, serine, inositol, and glycerol, will expand the research about the integration mechanisms in antimicrobial peptide. CONCLUSIONS A spontaneous and oriented integration of temporin L into a BMP membrane was observed. The basic amino acids in temporin L played an important role in the integration into BMPs. The N-terminus of temporin L was located on a BMP membrane surface. The conjugation of biotin to the N-terminus of temporin L lead to binding of streptavidin on a BMP membrane. Furthermore, a complex of streptavidin and biotin binding to the N-terminus of temporin L-lysine was successfully assembled onto BMPs. Temporin L will be a powerful tool that allows a quick and spontaneous integration. ACKNOWLEDGMENT This work was funded in part by a Grant-in-Aid for Specially Promoted Research, No. 13002005 and Young Scientists (B), No. 15760583 from the Japan Society for the Promotion of Science.

Received for review November 18, 2003. Accepted March 25, 2004. AC035361M

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